| BackgroundBrain metabolism has its own characteristics, cerebral oxygen consumption is highest, the glucose metabolism of brain is the most productive, but sugar is the main substance for the brain’s energy metabolism, so the brain’s energy and metabolic substrate is very few, the stored energy is rare. Therefore, cardiac arrest, severe shock and cerebral infarction can affect the whole brain or local cerebral blood circulation, the blood flow to the brain suddenly reduces or stops, greatly reducing the oxygen and brain glucose supply, causes further cerebral cellular energy failure, which may cause serious ischemic neuronal injury and irreversible brain injury at last, especially in the susceptable hippocampus. Finally, it will result in neurological impairment, cognitive impairment, such as memory and sensorimotor disorder. Approximately 326,000 ambulatory and 209,000 hospitalized patients experience cardiac arrest (CA) annually in the United States. Unfortunately, despite regular updates of cardiopulmonary resuscitation guidelines, treatment outcomes of CA remain poor, with less than 9% of cardiac arrest patients reported as surviving with good neurological function. Transient global cerebral ischemia (GCI) induces ischemic reperfusion (I/R) injury, and brain injury is the predominant cause of high morbidity and mortality of CA and resuscitation. which affects the quality of life and brings heavy economic burden to the family. Consequently, this kind of injury endangers seriously human health, But there is still lack of effective treatment. Although there are some neuroprotective agents against acute cerebral ischemia or ischemia/reperfusion injury following reperfusion, its efficacy and safety is still uncertain.The mechanism of global cerebral ischemia and reperfusion injury is not clear. Inflammation is important host response to injury. Studies have shown that neuro-inf lammation plays a key role in many diseases of the central nervous system, including global cerebral ischemic injury, proinflammatory cytokine such as interleukin 1 beta and interleukin 18, is considered to be important mediators of neuro-inf lammation, but they need to be converted from an inactive precursor proteininto active form, interleukin-1β-converting enzyme (ICE), also known as caspases-1, therefore, caspase-1 is considered to be synonymous with inflammation, however, caspase-1 plays important role in brain ischemia by promoting both apoptotic and inflammatory processes. As reported in the literature, caspase-1 can be directly involved in the noninfectious cell death process. In knockout caspase-1 mice or inhibiting caspase-1, it can reduce the brain damage caused by cerebral ischemia and improve the neurological function after ischemia. Therefore, caspase-1 inhibitors may play a neuroprotective role in ischemic brain injury. If we prove that the caspase-1 inhibitors is effective in the treatment of ischemic brain injury and then, we explore the possible mechanism, which can provide new clues and potential drug targets in the treatment of ischemic brain injury, this research has important medical value and social significance.Caspase-1 is an enzyme implicated in neuro-inflammation, a critical component of many diseases that affect the central nervous system. Intriguingly, caspase-1 is not only regarded as a key mediator of inflammatory processes but also as a key executioner of apoptosis, and increasing evidence suggests that neuro-inflammation plays a pivotal role in the neuronal cell death that occurs following cerebral ischemia.Objective1. to assess whether the caspase-1 inhibitor Boc-D-CMK attenuates neuro-inflammation in the hippocampus following transient global cerebral ischemia.2. To verify whether intranasal administration of a caspase-1 inhibitor can attenuate apoptotic neuronal damage and hippocampal CA1 neuronal injury induced by transient global cerebral ischemia.3. To study whether intranasal administration of a caspase-1 inhibitor can ameliorate functional impairment following transient global cerebral ischemia.4. To explore the protective mechanism of intranasal administration of a caspase-1 inhibitor on transient global cerebral ischemia.Methods1. Male clean 6 Sprague Dawley (SD) rats, Biotin-labeled caspase 1 inhibitor was delivered intranasally to a subset of SD rats in order to observe hippocampal drug’s distribution and localization in brain. This subset of rats was sacrificed 12 h after drug administration.2. Male clean 12 SD rats, In the 4-vessel occlusion rat model of global cerebral ischemia, the activity of Caspase-1 was detected by Spectrophotometry after ischemia reperfusion.3. Male clean 48 SD rats were randomly divided into three groups:(a) Sham control group, (b) GCI group with vehicle infusion, and (3) GCI group with Boc-D-CMK treatment. In the 4-vessel occlusion rat model of global cerebral ischemia, Westernblot and immunohistochemical were used to examine protein levels, the activity of enzyme was detected by Spectrophotometry, ELISA was used to detect the secretion of inflammatory factors, Immunofluorescence, immunochemistry, confocal microscopy were used to examine the interactions between proteins, mitochondrial membrane potential, the survival and apoptosis neurons in pyramidal cell layer of the hippocamal CA1. Barnes Maze and Novel Object Rocognition Test were used to observe functional impairment of rats.Results1. Characterization of caspase-1 protein expression in hippocampal CA1 region of adult male SD rats following GCI.First, we determined the basal expression of caspase-1 protein in the hippocampal CA1 region of sham-operated rats, which do not undergo ischemia. Immunofluorescence staining for caspase-1 (Casp 1, green), MAP2 (red) and GFAP (red) revealed high levels of immunoreactive caspase-1 staining in the hippocampal CA1 region. Intriguingly, caspase-1 immunostaining was found to co-localize both with the neuronal marker MAP2 and the astrocyte marker GFAP, suggesting that caspase-1 is expressed in both neurons and astrocytes in the rat brain at baseline. We next performed immunofluorescence staining for the same markers in adult rats subjected to GCI. Compared to the basal distribution of caspase-1 immunostaining in the hippocampal CA1 region seen in sham-operated animals, confocal analysis demonstrated statistically significant overexpression of caspase-1 protein in astrocytes both 3 days and 14 days following GCI.2. Neural distribution of biotin-labeled caspase-1 inhibitor after intranasal administration.a subset of rats were sacrificed at 12 h after intranasal delivery of a biotin-labeled caspase-1 inhibitor. A Streptavidin Alexa Fluor 488 conjugated antibody (Thermo Fisher Scientific Inc.) was then used for confocal microscopy to visualize the caspase-1 inhibitor within the brain, The Biotin-labeled caspase-1 inhibitor successfully penetrated the rat brain after intranasal administration, with a significant portion reaching the ischemia-vulnerable hippocampal CA1 region. Furthermore, confocal microscopy showed that the intranasally delivered caspase-1 inhibitor mainly localized to the cellular cytoplasm of neurons in the hippocampal CA1 and the cortex. This serves as poof of concept that a caspase-1 inhibitor can penetrate the central nervous system via the nasal cavity, effectively circumventing the blood-brain barrier through intranasal delivery.3. Intranasal Boc-D-GMK post-treatment inhibits GCI-induced caspase-1 overexpression, cleavage, and activity in hippocampal CA1 region.In order to determine whether intranasal Boc-D-CMK post-treatment can exert neuroprotective effects in a GCI rat model, we initially measured the time course of caspase-1 activity in the hippocampal CA1 region after transient GCI using a f luorometric substrate assay. Compared to sham-operated animals, caspase-1 activity was increased at all time points observed after ischemia, reaching a peak level about at 3 days post GCI and remaining at a high level at 5 days post GCI. Confocal microscopy revealed that the cleaved-caspase-1 (active form) was markedly increased and co-localized to astrocytes at 3 days post GCI compared with sham controls. By contract, the expression and localization of cleaved-caspase-1 were drastically decreased in Boc-D-CMK treated animals. Importantly, intranasal Boc-D-CMK post-treatment significantly decreased the activity of caspase-1 in hippocampal CA1 region at 3 days following GCI, in comparison with the I/R control group. Western blotting demonstrated that the expression of caspase-1 and cleaved-caspase-1 were both significantly increased in the hippocampal CA1 region at 3 days after GCI, and the high-level expression were effectively suppressed by intranasal Boc-D-CMK treatment.4. Intranasal Boc-D-CMK post-treatment improves GCI-induced mitochondrial dysfunction in hippocampal GA1 region.In the present study, intranasal Boc-D-CMK post-treatment reversed the GCI-induced change in Mitotracker Red fluorescence intensity, intranasal Boc-D-CMK post-treatment significantly increased cytochrome c oxidase activity of mitochondria, relative to I/R group. Western blotting and data analyses further indicated that Boc-D-CMK attenuated GCI-induced release of mitochondrial cytochrome c, as evidenced by decreased cytosolic level of cytochrome c and increased mitochondrial level of cytochrome c at 2 days after GCI. Taken together, these data suggest that intranasal Boc-D-CMK post-treatment has the ability to preserve MMP and rescue GCI-induced mitochondrial dysfunction in CA1 region following I/R.5. Intranasal Boc-D-CMK post-treatment suppresses caspase-9 and caspase-3 activation in hippocampal CA1 region.The data has shown that 3-day I/R evoked significant increases in the activities of caspase-9 and caspse-3. Importantly, the increased activities were dramatically prevented in Boc-D-CMK-treated animals. In addition, the expressions and localizations of cleaved-caspase-3 and cleaved-caspase-9 were examined by double immunofluorescence staining with NeuN on the brain sections 3 days after I/R. Representative confocal microscopy imaging and analysis certified that, there were significant increases in cleaved-caspase-3 and cleaved-caspase-9 immunostainings compared to sham, which co-localized with NeuN-positive cells, in the I/R group. Meaningfully, Boc-D-CMK post-treatment markedly attenuated the neuronal localizations of cleaved-caspase-9 and cleaved-caspase-3 compared to I/R control group.6. Intranasal Boc-D-CMK post-treatment inhibits the intrinsic apoptotic pathway in hippocampal CA1 region following GCI.Immunofluorescence staining indicated that,3-day ischemic reperfusion induced remarkable TUNEL and PARP1 positive staining in the hippocampal CA1 cell layer, compared with sham and Boc-D-CMK treated groups. Quantitative analysis showed that the number of TUNEL-positive and PARP1-positive cells in the CA1 pyramidal cell layer was significantly attenuated in the Boc-D-CMK post-treated animals compared with the I/R animals, indicating that Boc-D-CMK has the ability to inhibit the intrinsic apoptotic neuronal death following GCI.7. Intranasal Boc-D-CMK post-treatment attenuates GCI-induced microglial activation and astrocytosis following GCI in hippocampal CA1 regionwe first examined the glial activation at the early stage following ischemic reperfusion. Western blotting and quantification analyses of Ibal and GFAP in total protein samples from hippocampal CA1 region showed that the Ibal and GFAP levels were significantly increased compared with sham control groups at 3 days after GCI, suggesting GCI-induced reactive gliosis. Interestingly, Boc-D-CMK treated animals exhibited robust decreases in the levels of Ibal and GFAP versus I/R control group. In order to analyze microglial and astrocyte activation occurring during the late phase of cerebral injury, the brain sections 14 days after GCI were selected for Ibal and GFAP staining. Immunofluorescence staining and relative intensity analyses indicated that the microglial activation and astrocytosis was dramatically enhanced at 14 days post GCI in rat hippocampal CA1 region, compared to the sham group. As excepted, GCI-induced reactive gliosis was significantly reduced in the Boc-D-CMK treatment group, compared to the I/R group 14 days post GCI.8. Intranasal Boc-D-CMK post-treatment inhibits GCI-induced production of pro-inflammatory cytokine and prevents GCI-induced delayed neuronal cell death in hippocampal CA1 region.The levels of IL-1β,IL-18,IL-6 and TNF-α were all increased significantly in the ischemic control group compared to sham animals. However, intranasal Boc-D-CMK post-treatment markedly reduced the secretion of all inflammatory cytokines tested. These data suggest that the caspase-1 inhibitor Boc-D-CMK attenuated neuro-inflammation in the CA1 hippocampal region following GCI by preventing maturation of inflammatory cytokines. Histological evaluation via Cresyl Violet staining and NeuN staining confirmed that transient GCI induced profound local neuronal loss in the ischemia-vulnerable hippocampal CA1 region in the I/R animals, compared to sham groups. Cresyl Violet-stained sections from animals of the I/R group showed unequivocal signs of cell death 14 days after GCI, as the pyramidal cells had condensed, pyknotic, and shrunken nuclei. In contrast, histology and quantitative analysis showed that intranasal Boc-D-CMK post-treatment significantly increased the number of surviving neurons in the hippocampal CA1 region 14 days following GCI, compared to the non-treated I/R group.9. Boc-D-CMK post-treatment attenuates GCI-induced spatial learning and memory deficits and enhances recognition memory following GCI in Novel Object Recognition test.The I/R animals took more time to locate the black escape box during training trials at 10 days after GCI, compared to sham-operated animals. Interestingly, Boc-D-CMK treated animals significantly decreased escape latencies to find the escape box on the final day of training trials. In the probe test on day 11 after GCI, the I/R animals spent significantly less time in the target quadrant (where the escape box had been) than the Boc-D-CMK-treated animals and the sham animals. These data suggest that the caspase-1 ihibitor Boc-D-CMK has the ability to significantly attenuate long-term learning and memory impairment following GCI. Fruthermore, the hippocampal integrity is necessary for non-spatial tasks, such as object recognition memory. The NOR task was performed as described in Methods to test the long-term recognition memory of the animals in each group, the recognition index in the Choice session on day 13 was significantly decreased in the I/R control group and the decrease was effectively reversed in the Boc-D-CMK-treated animals after I/R, we certified intranasal Boc-D-CMK post-treatment would improve recognition deficits induced by I/R after intransient GCI.Conelusions1. we show that intranasal Boc-D-CMK readily penetrated the central nervous system, subsequently inhibiting caspase-1 activity, decreasing mitochondrial dysfunction, and attenuating caspase-3 dependent apoptotic pathway in ischemia-vulnerable hippocampal CA1 region.2. Correspondingly, behavioral tests showed that deteriorations in spatial learning and memory performance, and long-term recognition memory following GCI were significantly improved in the Boc-D-CMK post-treated animals.3. Further investigation regarding the mechanisms underlying Boc-D-CMK’s neuroprotective effects, revealed marked inhibition of reactive gliosis, as well as reduction of the neuro-inflammatory response via inhibition of the downstream pro-inflammatory cytokines production. Intranasal Boc-D-CMK post-treatment also significantly enhanced the numbers of NeuN-positive cells while simultaneously decreasing the numbers of TUNEL-positive and PARP1-positive cells in hippocampal CA1.In summary, the current study demonstrates that the caspase-1 inhibitor Boc-D-CMK coordinates anti-inflammatory and anti-apoptotic actions to attenuate neuronal death in the hippocampal CA1 region following GCI. Furthermore, our data suggest that pharmacological inhibition of caspase-1 is a promising neuroprotective strategy to target ischemic neuronal injury and functional deficits following transient GCI. |
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